Cytotoxicity Mediation of Cells Evidencing Surface Expression of CD44

ABSTRACT

This invention relates to the diagnosis and treatment of cancerous diseases, particularly to the mediation of cytotoxicity of primary and metastatic human tumor cells; and most particularly to the use of an isolated monoclonal antibody or cancerous disease modifying antibodies (CDMAB) thereof, optionally in combination with one or more chemotherapeutic agents, as a means for initiating the cytotoxic response in such human tumors, e.g. any primary or metastatic tumor sites which arise from hepatocytes. The invention further relates to binding assays which utilize the CDMAB of the instant invention.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part to U.S. patent applicationSer. No. 11/716,216, filed on Mar. 9, 2007, which is acontinuation-in-part to U.S. patent application Ser. No. 11/364,013,filed on Feb. 28, 2006, which is a continuation-in-part to U.S. patentapplication Ser. No. 10/810,165, filed Mar. 26, 2004, now abandoned,which is a continuation-in-part to U.S. patent application Ser. No.10/647,818, filed Aug. 22, 2003, which is a continuation-in-part to U.S.patent application Ser. No. 10/603,000, filed Jun. 23, 2003, which is acontinuation-in-part to U.S. patent application Ser. No. 09/727,361 nowU.S. Pat. No. 6,657,048, issued Dec. 2, 2003, which is acontinuation-in-part to U.S. patent application Ser. No. 09/415,278 nowU.S. Pat. No. 6,180,357, issued Jan. 30, 2001, the contents of each ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the isolation and production of cancerousdisease modifying antibodies (CDMAB) and to the use of these CDMAB aloneor in combination with one or more CDMAB/chemotherapeutic agents intherapeutic and diagnostic processes. The invention further relates tobinding assays which utilize the CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

CD44 in Cancer: Raising monoclonal antibodies against human white bloodcells led to the discovery of the CD44 antigen; a single chainhyaluronic acid (HA) binding glycoprotein expressed on a wide variety ofnormal tissue and on all types of hematopoietic cells. It was originallyassociated with lymphocyte activation and homing. Currently, itsputative physiological role also includes activation of inflammatorygenes, modulation of cell cycle, induction of cell proliferation,induction of differentiation and development, induction of cytoskeletalreorganization and cell migration and cell survival/resistance toapoptosis.

In humans, the single gene copy of CD44 is located on the short arm ofchromosome 11, 11p13. The gene contains 19 exons; the first 5 areconstant, the next 9 are variant, the following 3 are constant and thefinal 2 are variant. Differential splicing can lead to over 1000different isoforms. However, currently only several dozen naturallyoccurring variants have been identified.

The CD44 standard glycoprotein consists of a N-terminal extracellular(including a 20 a.a. leader sequence, and a membrane proximal region (85a.a.)) domain (270 a.a.), a transmembrane region (21 a.a.) and acytoplasmic tail (72 a.a.). The extracellular region also contains alink module at the N-terminus. This region is 92 a.a. in length andshows homology to other HA binding link proteins. There is high homologybetween the mouse and human forms of CD44. The variant forms of theprotein are inserted to the carboxy terminus of exon 5 and are locatedextracellularly when expressed.

A serum soluble form of CD44 also occurs naturally and can arise fromeither a stop codon (within the variable region) or from proteolyticactivity. Activation of cells from a variety of stimuli including TNF-αresults in shedding of the CD44 receptor. Shedding of the receptor hasalso been seen with tumor cells and can result in an increase in thehuman serum concentration of CD44 by up to 10-fold. High CD44 serumconcentration suggests malignancy (ovarian cancer being the exception).

The standard form of CD44 exists with a molecular weight ofapproximately 37 kD. Post-translational modifications increase themolecular weight to 80-90 kD. These modifications include amino terminusextracellular domain N-linked glycosylations at asparagine residues,O-linked glycosylations at serine/threonine residues at the carboxyterminus of the extracellular domain and glycosaminoglycan additions.Splice variants can range in size from 80-250 kD.

HA, a polysaccharide located on the extracellular matrix (ECM) inmammals, is thought to be the primary CD44 ligand. However, CD44 hasalso been found to bind such proteins as collagen, fibronectin, lamininetc. There appears to be a correlation between HA binding andglycosylation. Inactive CD44 (does not bind HA) has the highest levelsof glycosylation, active CD44 (binding HA) the lowest while inducibleCD44 (does not or weakly binds HA unless activated by cytokines,monoclonal antibodies, growth factors, etc.) has glycosylation levelssomewhere in between the active and inactive forms.

CD44 can mediate some of its functions through signal transductionpathways that depend on the interaction of the cell, stimulus and theenvironment. Some of these pathways include the NFκB signaling cascade(involved in the inflammatory response), the Ras-MAPK signaltransduction pathway (involved with activating cell cycling andproliferation), the Rho family of proteins (involved with cytoskeletonreorganization and cell migration) and the PI3-K-related signalingpathway (related to cell survival). All of the above-mentioned functionsare closely associated with tumor disease initiation and progression.CD44 has also been implicated in playing a role in cancer through avariety of additional mechanisms. These include the presentation ofgrowth factors, chemokines and cytokines by cell surface proteoglycanspresent on the cell surface of CD44 to receptors involved in malignancy.Also, the intracellular degradation of HA by lysosomal hyaluronidasesafter internalization of the CD44-HA complex can potentially increasethe likelihood of tumor invasiveness and induction of angiogenesisthrough the ECM. In addition, the transmission of survival or apoptoticsignals has been shown to occur through either the standard or variableCD44 receptor. CD44 has also been suggested to be involved in celldifferentiation and migration. Many, if not all, of these mechanisms areenvironment and cell dependent and several give rise to variablefindings. Therefore, more research is required before any conclusionscan be drawn.

In order to validate a potential functional role of CD44 in cancer,expression studies of CD44 were undertaken to determine if differentialexpression of the receptor correlates with disease progression. However,inconsistent findings were observed in a majority of tumor types andthis is probably due to a combination of reagents, technique,pathological scoring and cell type differences between researchers.Renal cell carcinoma and non-Hodgkin's lymphoma appear to be theexception in that patients with high CD44 expressing tumors consistentlyhad shorter survival times than their low or non-CD44 expressingcounterparts.

Due to its association with cancer, CD44 has been the target of thedevelopment of anti-cancer therapeutics. There is still controversy asto whether the standard or the variant forms of CD44 are required fortumor progression. There is in vivo animal data to support both viewsand again it may be tumor type and even cell type dependent. Differenttherapeutic approaches have included injection of soluble CD44 proteins,hyaluronan synthase cDNA, hyaluronidase, the use of CD44 antisense andCD44 specific antibodies. Each approach has led to some degree ofsuccess thereby providing support for anti-CD44 cancer therapeutics.

Both variant and standard CD44 specific monoclonal antibodies have beengenerated experimentally but for the most part these antibodies have nointrinsic biological activity, rather they bind specifically to the typeof CD44 they recognize. However, there are some that are either activein vitro or in vivo but generally not both. Several anti-CD44 antibodieshave been shown to mediate cellular events. For example the murineantibody A3D8, directed against human erythrocyte Lutheran antigen CD44standard form, was shown to enhance CD2 (9-1 antibody) and CD3 (OKT3antibody) mediated T cell activation; another anti-CD44 antibody hadsimilar effects. A3D8 also induced IL-1 release from monocytes and IL-2release from T lymphocytes. Interestingly, the use of A3D8 inconjunction with drugs such as daunorubicin, mitoxantrone and etoposideinhibited apoptosis induction in HL60 and NB4 AML cells by abrogatingthe generation of the second messenger ceramide. The J173 antibody,which does not have intrinsic activity and is directed against a similarepitope of CD44s, did not inhibit drug-induced apoptosis. The NIH44-1antibody, directed against an 85-110 kD and 200 kD form of CD44,augmented T-cell proliferation through a pathway the authors speculatedas either cross-linking or aggregation of CD44. Taken together, there isno evidence that antibodies such as these are suitable for use as cancertherapeutics since they either are not directed against cancer (e.g.activate lymphocytes), induce cell proliferation, or when used withcytotoxic agents inhibited drug-induced death of cancer cells.

Several anti-CD44 antibodies have been described which demonstrateanti-tumor effects in vivo. The antibody 1.1 ASML, a mouse IgG1 directedto the v6 variant of CD44, has been shown to decrease the lymph node andlung metastases of the rat pancreatic adenocarcinoma BSp73ASML. Survivalof the treated animals was concomitantly increased. The antibody wasonly effective if administered before lymph node colonization, and waspostulated to interfere with cell proliferation in the lymph node. Therewas no direct cytototoxicity of the antibody on the tumor cells invitro, and the antibody did not enhance complement-mediatedcytotoxicity, or immune effector cell function. Utility of the antibodyagainst human cells was not described.

Breyer et al. described the use of a commercially-available antibody toCD44s to disrupt the progression of an orthotopically-implanted ratglioblastoma. The rat glioblastoma cell line C6 was implanted in thefrontal lobe, and after 1 week, the rats were given 3 treatments withantibody by intracerebral injection. Treated rats demonstrated decreasedtumor growth, and higher body weight than buffer or isotype controltreated rats. The antibody was able to inhibit adhesion of cells invitro to coverslips coated with extracellular matrix components, but didnot have any direct cytotoxic effects on cells. This antibody was nottested against human cells.

A study was carried out which compared the efficacy of an antibody toCD44s (IM-7.8.1) to an antibody to CD44v10 (K926). The highly metastaticmurine melanoma line B16F10, which expresses both CD44 isoforms, wasimplanted intravenously into mice. After 2 days, antibodies were givenevery third day for the duration of the study. Both antibodies caused asignificant reduction of greater than 50 percent in the number of lungmetastases; there was no significant difference in efficacy between thetwo antibodies. The antibody did not affect proliferation in vitro, andthe authors, Zawadzki et al., speculated that the inhibition of tumorgrowth was due to the antibody blocking the interaction of CD44 with itsligand. In another study using IM-7.8.1, Zahalka et al. demonstratedthat the antibody and its F(ab′)₂ fragment were able to block the lymphnode infiltration by the murine T-cell lymphoma LB. This conferred asignificant survival benefit to the mice. Wallach-Dayan et al. showedthat transfection of LB-TRs murine lymphoma, which does notspontaneously form tumors, with CD44v4-v10 conferred the ability to formtumors. IM-7.8.1 administration decreased tumor size of the implantedtransfected cells in comparison to the isotype control antibody. None ofthese studies demonstrated human utility for this antibody.

GKW.A3, a mouse IgG2a, is specific for human CD44 and prevents theformation and metastases of a human melanoma xenograft in SCID mice. Theantibody was mixed with the metastastic human cell line SMMU-2, and theninjected subcutaneously. Treatments were continued for the following 3weeks. After 4 weeks, only 1 of 10 mice developed a tumor at theinjection site, compared to 100 percent of untreated animals. F(ab′)₂fragments of the antibody demonstrated the same inhibition of tumorformation, suggesting that the mechanism of action was not dependent oncomplement or antibody-dependent cellular cytotoxicity. If the tumorcells were injected one week prior to the first antibody injection, 80percent of the animals developed tumors at the primary site. However, itwas noted that the survival time was still significantly increased.Although the delayed antibody administration had no effect on theprimary tumor formation, it completely prevented the metastases to thelung, kidney, adrenal gland, liver and peritoneum that were present inthe untreated animals. This antibody does not have any directcytotoxicity on the cell line in vitro nor does it interfere withproliferation of SMMU-2 cells, and appears to have its major effect ontumor formation by affecting metastasis or growth. One notable featureof this antibody was that it recognized all isoforms of CD44, whichsuggests limited possibilities for therapeutic use.

Strobel et al. describe the use of an anti-CD44 antibody (clone 515) toinhibit the peritoneal implantation of human ovarian cancer cells in amouse xenograft model. The human ovarian cell line 36M2 was implantedintraperitoneally into mice in the presence of the anti-CD44 antibody orcontrol antibody, and then treatments were administered over the next 20days. After 5 weeks, there were significantly fewer nodules in theperitoneal cavity in the antibody treated group. The nodules from boththe anti-CD44 and control treated groups were the same size, suggestingthat once the cells had implanted, the antibody had no effect on tumorgrowth. When cells were implanted subcutaneously there was also noeffect on tumor growth indicating that the antibody itself did not havean anti-proliferative or cytotoxic effect. In addition, there was noeffect of the antibody on cell growth in vitro.

VFF-18, also designated as BIWA 1, is a high-affinity antibody to the v6variant of CD44 specific for the 360-370 region of the polypeptide. Thisantibody has been used as a ^(99m)Technetium-labelled conjugate in aPhase 1 clinical trial in 12 patients. The antibody was tested forsafety and targeting potential in patients with squamous cell carcinomaof the head and neck. Forty hours after injection, 14 percent of theinjected dose was taken up by the tumor, with minimal accumulation inother organs including the kidney, spleen and bone marrow. The highlyselective tumor binding suggests a role for this antibody inradioimmunotherapy, although the exceptionally high affinity of thisantibody prevented penetration into the deeper layers of the tumor.Further limiting the application of BIWA 1 is the immunogenicity of themurine antibody (11 of 12 patients developed human anti-mouse antibodies(HAMA)), heterogenous accumulation throughout the tumor and formation ofantibody-soluble CD44 complexes. WO 02/094879 discloses a humanizedversion of VFF-18 designed to overcome the HAMA response, designatedBIWA 4. BIWA 4 was found to have a significantly lower antigen bindingaffinity than the parent VFF 18 antibody. Surprisingly, the loweraffinity BIWA 4 antibody had superior tumor uptake characteristics thanthe higher affinity BIWA 8 humanized VFF-18 antibody. Both^(99m)Technetium-labelled and ¹⁸⁶Rhenium-labelled BIWA 4 antibodies wereassessed in a 33 patient Phase 1 clinical trial to determine safety,tolerability, tumor accumulation and maximum tolerated dose, in the caseof ¹⁸⁶Re-labelled BIWA 4. There appeared to be tumor related uptake of^(99m)Tc-labelled BIWA 4. There were no tumor responses seen with alldoses of ¹⁸⁶Re-labelled BIWA 4, although a number had stable disease;the dose limiting toxicity occurred at 60 mCi/m². There was a 50-65percent rate of adverse events with 12 of 33 patients deemed to haveserious adverse events (thrombocytopenia, leukopenia and fever) and ofthose 6, all treated with ¹⁸⁶Re-labelled BIWA 4, died in the course oftreatment or follow-up due to disease progression. Two patientsdeveloped human anti-human antibodies (HAHA). A Phase 1 dose escalationtrial of ¹⁸⁶Re-labelled BIWA 4 was carried out in 20 patients. Oralmucositis and dose-limiting thrombocytopenia and leucocytopenia wereobserved; one patient developed a HAHA response. Stable disease was seenin 5 patients treated at the highest dose of 60 mCi/m². Although deemedto be acceptable in both safety and tolerability for the efficacyachieved, these studies have higher rates of adverse events compared toother non-radioisotope conjugated biological therapies in clinicalstudies. U.S. Patent Application US 2003/0103985 discloses a humanizedversion of VFF-18 conjugated to a maytansinoid, designated BIWI 1, foruse in tumor therapy. A humanized VFF 18 antibody, BIWA 4, whenconjugated to a toxin, i.e. BIWI 1, was found to have significantanti-tumor effects in mouse models of human epidermoid carcinoma of thevulva, squamous cell carcinoma of the pharynx or breast carcinoma. Theunconjugated version, BIWA 4, did not have anti-tumor effects and theconjugated version, BIWI 1, has no evidence of safety or efficacy inhumans.

Mab U36 is a murine monoclonal IgG1 antibody generated by UM-SCC-22Bhuman hypopharyngeal carcinoma cell immunization and selection forcancer and tissue specificity. Antigen characterization through cDNAcloning and sequence analysis identified the v6 domain ofkeratinocyte-specific CD44 splice variant epican as the target of MabU36. Immunohistochemistry studies show the epitope to be restricted tothe cell membrane. Furthermore, Mab U36 labeled 94 percent of the headand neck squamous cell carcinomas (HNSCC) strongly, and within thesetumors there was uniformity in cell staining. A 10 patient^(99m)Tc-labelled Mab U36 study showed selective accumulation of theantibody to HNSCC cancers (20.4+/−12.4 percent injected dose/kg at 2days); no adverse effects were reported but two patients developed HAMA.In a study of radio-iodinated murine Mab U36 there were 3 cases of HAMAin 18 patients and selective homogenous uptake in HNSCC. In order todecrease the antigenicity of Mab U36 and decrease the rate of HAMA achimeric antibody was constructed. Neither the chimeric nor the originalmurine Mab U36 has ADCC activity. There is no evidence of nativefunctional activity of Mab U36. ¹⁸⁶Re-labelled chimeric Mab U36 was usedto determine the utility of Mab U36 as a therapeutic agent. In thisPhase 1 escalating dose trial 13 patients received a scouting dose of^(99m)Tc-labelled chimeric Mab U36 followed by ¹⁸⁶Re-labelled chimericMab U36. There were no acute adverse events reported but followingtreatment dose limiting myelotoxcity (1.5 GBq/m²) in 2 of 3 patients,and thrombocytopenia in one patient treated with the maximum tolerateddose (1.0 GBq/m²) were observed. Although there were some effects ontumor size these effects did not fulfill the criteria for objectiveresponses to treatment. A further study of ¹⁸⁶Re-labelled chimeric MabU36 employed a strategy of using granulocyte colony-stimulating factorstimulated whole blood reinfusion to double the maximum-toleratedactivity to 2.8 Gy. In this study of nine patients with various tumorsof the head and neck, 3 required transfusions for drug related anemia.Other toxicity includes grade 3 myelotoxicity, and grade 2 mucositis. Noobjective tumor responses were reported although stable disease wasachieved for 3-5 months in 5 patients. Thus, it can be seen thatalthough Mab U36 is a highly specific antibody the disadvantage ofrequiring a radioimmunoconjugate to achieve anti-cancer effects limitsits usefulness because of the toxicity associated with the therapy inrelation to the clinical effects achieved.

To summarize, a CD44v6 (1.1ASML) and CD44v10 (K926) monoclonal antibodyhave been shown to reduce metastatic activity in rats injected with ametastatic pancreatic adenocarcinoma or mice injected with a malignantmelanoma respectively. Another anti-CD44v6 antibody (VFF-18 and itsderivatives), only when conjugated to a maytansinoid or a radioisotope,has been shown to have anti-tumor effects. Anti-standard CD44 monoclonalantibodies have also been shown to suppress intracerebral progression byrat glioblastoma (anti-CD44s), lymph node invasion by mouse T celllymphoma (IM-7.8.1) as well as inhibit implantation of a human ovariancancer cell line in nude mice (clone 515), lung metastasis of a mousemelanoma cell line (IM-7.8.1) and metastasis of a human melanoma cellline in SCID mice (GKW.A3). The radioisotope conjugated Mab U36anti-CD44v6 antibody and its derivatives had anti-tumor activity inclinical trials that were accompanied by significant toxicity. Theseresults, though they are encouraging and support the development ofanti-CD44 monoclonal antibodies as potential cancer therapeutics,demonstrate limited effectiveness, safety, or applicability to humancancers.

Thus, if an antibody composition were isolated which mediated cancerouscell cytotoxicity, as a function of its attraction to cell surfaceexpression of CD44 on said cells, a valuable diagnostic and therapeuticprocedure would be realized.

Monoclonal Antibodies as Cancer Therapy: Each individual who presentswith cancer is unique and has a cancer that is as different from othercancers as that person's identity. Despite this, current therapy treatsall patients with the same type of cancer, at the same stage, in thesame way. At least 30 percent of these patients will fail the first linetherapy, thus leading to further rounds of treatment and the increasedprobability of treatment failure, metastases, and ultimately, death. Asuperior approach to treatment would be the customization of therapy forthe particular individual. The only current therapy which lends itselfto customization is surgery. Chemotherapy and radiation treatment cannotbe tailored to the patient, and surgery by itself, in most cases isinadequate for producing cures.

With the advent of monoclonal antibodies, the possibility of developingmethods for customized therapy became more realistic since each antibodycan be directed to a single epitope. Furthermore, it is possible toproduce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

Having recognized that a significant difference between cancerous andnormal cells is that cancerous cells contain antigens that are specificto transformed cells, the scientific community has long held thatmonoclonal antibodies can be designed to specifically target transformedcells by binding specifically to these cancer antigens; thus giving riseto the belief that monoclonal antibodies can serve as “Magic Bullets” toeliminate cancer cells. However, it is now widely recognized that nosingle monoclonal antibody can serve in all instances of cancer, andthat monoclonal antibodies can be deployed, as a class, as targetedcancer treatments. Monoclonal antibodies isolated in accordance with theteachings of the instantly disclosed invention have been shown to modifythe cancerous disease process in a manner which is beneficial to thepatient, for example by reducing the tumor burden, and will variously bereferred to herein as cancerous disease modifying antibodies (CDMAB) or“anti-cancer” antibodies.

At the present time, the cancer patient usually has few options oftreatment. The regimented approach to cancer therapy has producedimprovements in global survival and morbidity rates. However, to theparticular individual, these improved statistics do not necessarilycorrelate with an improvement in their personal situation.

Thus, if a methodology was put forth which enabled the practitioner totreat each tumor independently of other patients in the same cohort,this would permit the unique approach of tailoring therapy to just thatone person. Such a course of therapy would, ideally, increase the rateof cures, and produce better outcomes, thereby satisfying a long-feltneed.

Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remission or responses. Furthermore, there was a lack ofreproducibility and there was no additional benefit compared tochemotherapy. Solid tumors such as breast cancers, melanomas and renalcell carcinomas have also been treated with human blood, chimpanzeeserum, human plasma and horse serum with correspondingly unpredictableand ineffective results.

There have been many clinical trials of monoclonal antibodies for solidtumors. In the 1980s there were at least four clinical trials for humanbreast cancer which produced only one responder from at least 47patients using antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-Her2/neu antibody (Herceptin®) incombination with CISPLATIN. In this trial 37 patients were assessed forresponses of which about a quarter had a partial response rate and anadditional quarter had minor or stable disease progression. The mediantime to progression among the responders was 8.4 months with medianresponse duration of 5.3 months.

Herceptin® was approved in 1998 for first line use in combination withTaxol®. Clinical study results showed an increase in the median time todisease progression for those who received antibody therapy plus Taxol®(6.9 months) in comparison to the group that received Taxol® alone (3.0months). There was also a slight increase in median survival; 22 versus18 months for the Herceptin® plus Taxol® treatment arm versus the Taxol®treatment alone arm. In addition, there was an increase in the number ofboth complete (8 versus 2 percent) and partial responders (34 versus 15percent) in the antibody plus Taxol® combination group in comparison toTaxol® alone. However, treatment with Herceptin® and Taxol® led to ahigher incidence of cardiotoxicity in comparison to Taxol® treatmentalone (13 versus 1 percent respectively). Also, Herceptin® therapy wasonly effective for patients who over express (as determined throughimmunohistochemistry (IHC) analysis) the human epidermal growth factorreceptor 2 (Her2/neu), a receptor, which currently has no known functionor biologically important ligand; approximately 25 percent of patientswho have metastatic breast cancer. Therefore, there is still a largeunmet need for patients with breast cancer. Even those who can benefitfrom Herceptin® treatment would still require chemotherapy andconsequently would still have to deal with, at least to some degree, theside effects of this kind of treatment.

The clinical trials investigating colorectal cancer involve antibodiesagainst both glycoprotein and glycolipid targets. Antibodies such as17-1A, which has some specificity for adenocarcinomas, has undergonePhase 2 clinical trials in over 60 patients with only 1 patient having apartial response. In other trials, use of 17-1A produced only 1 completeresponse and 2 minor responses among 52 patients in protocols usingadditional cyclophosphamide. To date, Phase III clinical trials of 17-1Ahave not demonstrated improved efficacy as adjuvant therapy for stageIII colon cancer. The use of a humanized murine monoclonal antibodyinitially approved for imaging also did not produce tumor regression.

Only recently have there been any positive results from colorectalcancer clinical studies with the use of monoclonal antibodies. In 2004,ERBITUX® was approved for the second line treatment of patients withEGFR-expressing metastatic colorectal cancer who are refractory toirinotecan-based chemotherapy. Results from both a two-arm Phase IIclinical study and a single arm study showed that ERBITUX® incombination with irinotecan had a response rate of 23 and 15 percentrespectively with a median time to disease progression of 4.1 and 6.5months respectively. Results from the same two-arm Phase II clinicalstudy and another single arm study showed that treatment with ERBITUX®alone resulted in an 11 and 9 percent response rate respectively with amedian time to disease progression of 1.5 and 4.2 months respectively.

Consequently in both Switzerland and the United States, ERBITUX®treatment in combination with irinotecan, and in the United States,ERBITUX® treatment alone, has been approved as a second line treatmentof colon cancer patients who have failed first line irinotecan therapy.Therefore, like Herceptin®, treatment in Switzerland is only approved asa combination of monoclonal antibody and chemotherapy. In addition,treatment in both Switzerland and the US is only approved for patientsas a second line therapy. Also, in 2004, AVASTIN® was approved for usein combination with intravenous 5-fluorouracil-based chemotherapy as afirst line treatment of metastatic colorectal cancer. Phase III clinicalstudy results demonstrated a prolongation in the median survival ofpatients treated with AVASTIN® plus 5-fluorouracil compared to patientstreated with 5-fluorouracil alone (20 months versus 16 monthsrespectively). However, again like Herceptin® and ERBITUX®, treatment isonly approved as a combination of monoclonal antibody and chemotherapy.

There also continues to be poor results for lung, brain, ovarian,pancreatic, prostate, and stomach cancer. The most promising recentresults for non-small cell lung cancer came from a Phase II clinicaltrial where treatment involved a monoclonal antibody (SGN-15; dox-BR96,anti-Sialyl-LeX) conjugated to the cell-killing drug doxorubicin incombination with the chemotherapeutic agent TAXOTERE®. TAXOTERE® is theonly FDA approved chemotherapy for the second line treatment of lungcancer. Initial data indicate an improved overall survival compared toTAXOTERE® alone. Out of the 62 patients who were recruited for thestudy, two-thirds received SGN-15 in combination with TAXOTERE® whilethe remaining one-third received TAXOTERE® alone. For the patientsreceiving SGN-15 in combination with TAXOTERE®, median overall survivalwas 7.3 months in comparison to 5.9 months for patients receivingTAXOTERE® alone. Overall survival at 1 year and 18 months was 29 and 18percent respectively for patients receiving SNG-15 plus TAXOTERE®compared to 24 and 8 percent respectively for patients receivingTAXOTERE® alone. Further clinical trials are planned.

Preclinically, there has been some limited success in the use ofmonoclonal antibodies for melanoma. Very few of these antibodies havereached clinical trials and to date none have been approved ordemonstrated favorable results in Phase III clinical trials.

The discovery of new drugs to treat disease is hindered by the lack ofidentification of relevant targets among the products of 30,000 knowngenes that could contribute to disease pathogenesis. In oncologyresearch, potential drug targets are often selected simply due to thefact that they are over-expressed in tumor cells. Targets thusidentified are then screened for interaction with a multitude ofcompounds. In the case of potential antibody therapies, these candidatecompounds are usually derived from traditional methods of monoclonalantibody generation according to the fundamental principles laid down byKohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein).Spleen cells are collected from mice immunized with antigen (e.g. wholecells, cell fractions, purified antigen) and fused with immortalizedhybridoma partners. The resulting hybridomas are screened and selectedfor secretion of antibodies which bind most avidly to the target. Manytherapeutic and diagnostic antibodies directed against cancer cells,including Herceptin® and RITUXIMAB, have been produced using thesemethods and selected on the basis of their affinity. The flaws in thisstrategy are two-fold. Firstly, the choice of appropriate targets fortherapeutic or diagnostic antibody binding is limited by the paucity ofknowledge surrounding tissue specific carcinogenic processes and theresulting simplistic methods, such as selection by overexpression, bywhich these targets are identified. Secondly, the assumption that thedrug molecule that binds to the receptor with the greatest affinityusually has the highest probability for initiating or inhibiting asignal may not always be the case.

Despite some progress with the treatment of breast and colon cancer, theidentification and development of efficacious antibody therapies, eitheras single agents or co-treatments, have been inadequate for all types ofcancer.

Prior Patents:

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree.

U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas and not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for generating a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single-chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function istwo-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an antinuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes, which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree.

U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies, which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas and not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to anti-Her2 antibodies, which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for generating a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function is2-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an anti-nuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

U.S. Pat. No. 5,916,561 discloses a specific antibody, VFF-18, and itsvariants directed against the variant exon v6 of the CD44 gene. Thisantibody is an improvement over the comparator antibody in that itrecognizes a human CD44 v6 variant rather than a rat CD44 v6 variant. Inaddition this antibody discloses diagnostic assays for CD44 v6expression. There was no in vitro or in vivo function disclosed for thisantibody.

U.S. Pat. No. 5,616,468 discloses a monoclonal antibody, Var3.1, raisedagainst a synthetic peptide containing a sequence encoded by the humanexon 6A of the CD44 gene. Specifically this antibody does not bind tothe 90 kD form of human CD44 and is distinguished from the Hermes-3antibody. A method for detection of the v6 variant of CD44 is provided,as well as a method for screening and assaying for malignanttransformation based on this antigen. A method for screening forinflammatory disease based on detecting the antigen in serum is alsoprovided.

U.S. Pat. No. 5,879,898 discloses a specific antibody that binds to a129 bp exon of a human CD44 variant 6 that produces a 43 amino acidpeptide. The monoclonal antibody is produced by a number of hybridomacell lines: MAK<CD44>M-1.1.12, MAK<CD44>M-2.42.3, MAK<CD44>M-4.3.16. Theantibody is generated from a fusion protein that contains at least ahexapeptide of the novel CD44 v6 amino acid sequence. Further, there isa disclosure of an immunoassay for the detection of exon 6 variant thatcan be used as a cancer diagnostic. Significantly, there is no in vitroor in vivo function of this antibody disclosed.

U.S. Pat. No. 5,942,417 discloses a polynucleotide that encodes a CD44like polypeptide, and the method of making a recombinant protein usingthe polynucleotide and its variants. Antibodies are claimed to thesepolypeptides however there are no specific examples and there are nodeposited clones secreting such antibodies. Northern blots demonstratethe appearance of the polynucleotide in several types of tissues, butthere is no accompanying evidence that there is translation andexpression of this polynucleotide. Therefore, there is no evidence thatthere were antibodies to be made to the gene product of thispolynucleotide, that these antibodies would have either in vitro or invivo function, and whether they would be relevant to human cancerousdisease.

U.S. Pat. No. 5,885,575 discloses an antibody that reacts with a variantepitope of CD44 and methods of identifying the variant through the useof the antibody. The isolated polynucleotide encoding this variant wasisolated from rat cells, and the antibody, mAb1.1ASML, directed againstthis variant recognizes proteins of molecular weight 120 kD, 150 kD, 180kD, and 200 kD. The administration of monoclonal antibody 1.1ASMLdelayed the growth and metastases of rat BSp73ASML in isogenic rats.Significantly 1.1ASML does not recognize human tumors as demonstrated byits lack of reactivity, to LCLC97 human large-cell lung carcinoma. Ahuman homolog was isolated from LCLC97 but no equivalent antibodyrecognizing this homolog was produced. Thus, although an antibodyspecific to a variant of rat CD44 was produced and shown to affect thegrowth and metastasis of rat tumors there is no evidence for the effectthe this antibody against human tumors. More specifically the inventorspoint out that this antibody does not recognize human cancers.

SUMMARY OF THE INVENTION

This application utilizes methodology for producing patient specificanti-cancer antibodies taught in the U.S. Pat. No. 6,180,357 forisolating hybridoma cell lines which encode for cancerous diseasemodifying monoclonal antibodies. These antibodies can be madespecifically for one tumor and thus make possible the customization ofcancer therapy. Within the context of this application, anti-cancerantibodies having either cell-killing (cytotoxic) or cell-growthinhibiting (cytostatic) properties will hereafter be referred to ascytotoxic. These antibodies can be used in aid of staging and diagnosisof a cancer, and can be used to treat tumor metastases. These antibodiescan also be used for the prevention of cancer by way of prophylactictreatment. Unlike antibodies generated according to traditional drugdiscovery paradigms, antibodies generated in this way may targetmolecules and pathways not previously shown to be integral to the growthand/or survival of malignant tissue. Furthermore, the binding affinitiesof these antibodies are suited to requirements for initiation of thecytotoxic events that may not be amenable to stronger affinityinteractions. Also, it is within the purview of this invention toconjugate standard chemotherapeutic modalities, e.g. radionuclides, withthe CDMAB of the instant invention, thereby focusing the use of saidchemotherapeutics. The CDMAB can also be conjugated to toxins, cytotoxicmoieties, enzymes e.g. biotin conjugated enzymes, cytokines,interferons, target or reporter moieties or hematogenous cells, therebyforming an antibody conjugate. The CDMAB can be used alone or incombination with one or more CDMAB/chemotherapeutic agents.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies, and a panel of antibodies specific to the tumorcan be produced either using the methods outlined herein or through theuse of phage display libraries in conjunction with the screening methodsherein disclosed. All the antibodies generated will be added to thelibrary of anti-cancer antibodies since there is a possibility thatother tumors can bear some of the same epitopes as the one that is beingtreated. The antibodies produced according to this method may be usefulto treat cancerous disease in any number of patients who have cancersthat bind to these antibodies.

In addition to anti-cancer antibodies, the patient can elect to receivethe currently recommended therapies as part of a multi-modal regimen oftreatment. The fact that the antibodies isolated via the presentmethodology are relatively non-toxic to non-cancerous cells allows forcombinations of antibodies at high doses to be used, either alone, or inconjunction with conventional therapy. The high therapeutic index willalso permit re-treatment on a short time scale that should decrease thelikelihood of emergence of treatment resistant cells.

If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival and ananti-cancer antibody conjugated to red blood cells can be effectiveagainst in situ tumors as well. Alternatively, the antibodies may beconjugated to other hematogenous cells, e.g. lymphocytes, macrophages,monocytes, natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC). For example murine IgM and IgG2a antibodies canactivate human complement by binding the C-1 component of the complementsystem thereby activating the classical pathway of complement activationwhich can lead to tumor lysis. For human antibodies the most effectivecomplement activating antibodies are generally IgM and IgG1. Murineantibodies of the IgG2a and IgG3 isotype are effective at recruitingcytotoxic cells that have Fc receptors which will lead to cell killingby monocytes, macrophages, granulocytes and certain lymphocytes. Humanantibodies of both the IgG1 and IgG3 isotype mediate ADCC.

The cytotoxicity mediated through the Fc region requires the presence ofeffector cells, their corresponding receptors, or proteins e.g. NKcells, T cells and complement. In the absence of these effectormechanisms, the Fc portion of an antibody is inert. The Fc portion of anantibody may confer properties that affect the pharmacokinetics of anantibody in vivo, but in vitro this is not operative.

The cytotoxicity assays under which we test the antibodies do not haveany of the effector mechanisms present, and are carried out in vitro.These assays do not have effector cells (NK, Macrophages, or T-cells) orcomplement present. Since these assays are completely defined by what isadded together, each component can be characterized. The assays usedherein contain only target cells, media and sera. The target cells donot have effector functions since they are cancer cells or fibroblasts.Without exogenous cells which have effector function properties there isno cellular elements that have this function. The media does not containcomplement or any cells. The sera used to support the growth of thetarget cells do not have complement activity as disclosed by thevendors. Furthermore, in our own labs we have verified the absence ofcomplement activity in the sera used. Therefore, our work evidences thefact that the effects of the antibodies are due entirely to the effectsof the antigen binding which is mediated through the Fab. Effectively,the target cells are seeing and interacting with only the Fab, sincethey do not have receptors for the Fc. Although the hybridoma issecreting complete immunoglobulin which was tested with the targetcells, the only part of the immunoglobulin that interacts with the cellsare the Fab, which act as antigen binding fragments.

With respect to the instantly claimed antibodies and antigen bindingfragments, the application, as filed, has demonstrated cellularcytotoxicity as evidenced by the data in FIG. 1. As pointed out above,and as herein confirmed via objective evidence, this effect was entirelydue to binding by the Fab to the tumor cells.

Ample evidence exists in the art of antibodies mediating cytotoxicitydue to direct binding of the antibody to the target antigen independentof effector mechanisms recruited by the Fc. The best evidence for thisis in vitro experiments which do not have supplemental cells, orcomplement (to formally exclude those mechanisms). These types ofexperiments have been carried out with complete immunoglobulin, or withantigen binding fragments such as F(ab)′2 fragments. In these types ofexperiments, antibodies or antigen binding fragments can directly induceapoptosis of target cells such as in the case of anti-Her2 and anti-EGFRantibodies, both of which have been approved by the US FDA for marketingin cancer therapy.

Another possible mechanism of antibody mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are three additional mechanisms of antibody-mediated cancer cellkilling. The first is the use of antibodies as a vaccine to induce thebody to produce an immune response against the putative antigen thatresides on the cancer cell. The second is the use of antibodies totarget growth receptors and interfere with their function or to downregulate that receptor so that its function is effectively lost. Thethird is the effect of such antibodies on direct ligation of cellsurface moieties that may lead to direct cell death, such as ligation ofdeath receptors such as TRAIL R1 or TRAIL R2, or integrin molecules suchas alpha V beta 3 and the like.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematolgy 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end 2003, there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction, which may correlateto a delay in disease progression, extended survival or both, can alsolead to direct benefits and have clinical impact (Eckhardt et al.Developmental Therapeutics: Successes and Failures of Clinical TrialDesigns of Targeted Compounds; ASCO Educational Book, 39^(th) AnnualMeeting, 2003, pages 209-219).

Using substantially the process of U.S. Pat. No. 6,180,357, the mousemonoclonal antibody H460-16-2 was obtained following immunization ofmice with cells from a patient's lung tumor biopsy and from the lungcancer cell line NCI-H460 (ATCC, Virginia, MA). The H460-16-2 antigenwas expressed on the cell surface of a broad range of human cell linesfrom different tissue origins. The breast cancer cell line MDA-MB-231(MB-231) and skin cancer cell line A2058 were susceptible to thecytotoxic effects of H460-16-2 in vitro.

The result of H460-16-2 cytotoxicity against MB-231 cells in culture wasfurther extended by its anti-tumor activity towards these cancer cellswhen transplanted into mice (as disclosed in Ser. No. 10/603,000).Pre-clinical xenograft tumor models are considered valid predictors oftherapeutic efficacy.

In the preventative in vivo model of human breast cancer, H460-16-2treatment was significantly (p<0.0001) more effective in suppressingtumor growth during the treatment period than an isotype controlantibody. At the end of the treatment phase, mice given H460-16-2 hadtumors that grew to only 1.3 percent of the control group. During thepost treatment follow-up period, the treatment effects of H460-16-2 weresustained and the mean tumor volume in the treated groups continued tobe significantly smaller than controls until the end of the measurementphase. Using survival as a measure of antibody efficacy, it wasestimated that the risk of dying in the H460-16-2 treatment group wasabout 71 percent of the antibody buffer control group (p=0.028) at 70days post-treatment. These data demonstrated that H40-16-2 treatmentconferred a survival benefit compared to the control-treated groups.H460-16-2 treatment appeared safe, as it did not induce any signs oftoxicity, including reduced body weight and clinical distress. Thus,H460-16-2 treatment was efficacious as it both delayed tumor growth andenhanced survival compared to the control-treated groups in awell-established model of human breast cancer.

In addition, H460-16-2 demonstrated anti-tumor activity against MB-231cells in an established in vivo tumor model (as disclosed in Ser. No.10/603,000). Treatment with H460-16-2 was compared to the standardchemotherapeutic drug, Cisplatin, and it was shown that the Cisplatinand H460-16-2 treatment groups had significantly (p<0.001) smaller meantumor volumes compared with groups treated with either antibody dilutionbuffer or the isotype control antibody. H460-16-2 treatment mediatedtumor suppression that was approximately two-thirds that of Cisplatinchemotherapy but without the significant (19.2 percent) weight loss(p<0.003) and clinical distress, including 2 treatment-associateddeaths, observed with Cisplatin treatment. The anti-tumor activity ofH460-16-2 and its minimal toxicity make it an attractive anti-cancertherapeutic agent.

In the post-treatment period, H460-16-2 showed a significant survivalbenefit (p<0.02) as the risk of dying in the H460-16-2 group was abouthalf of that in the isotype control antibody group at >70 days aftertreatment. The observed survival benefit continued past 120 dayspost-treatment where 100 percent of the isotype control and Cisplatintreated mice had died compared to 67 percent of the H460-16-2 treatmentgroup. H460-16-2 maintained tumor suppression by delaying tumor growthby 26 percent compared to the isotype control antibody group. At 31 dayspost treatment, H460-16-2 limited tumor size by reducing tumor growth by48 percent compared to the isotype control group, which is comparable tothe 49 percent reduction observed at the end of the treatment. In theestablished tumor model of breast cancer, these results indicated thepotential of H460-16-2 to maintain tumor suppression beyond thetreatment phase and demonstrated the ability of the antibody to reducethe tumor burden and enhance survival in a mammal.

In addition to the beneficial effects in the established in vivo tumormodel of breast cancer, H460-16-2 treatment in combination with achemotherapeutic drug (Cisplatin) had anti-tumor activity against PC-3cells in an established in vivo prostate cancer model (as disclosed inSer. No. 10/810,165). Using a paired t-test, H460-16-2 plus Cisplatintreatment was significantly more effective in suppressing tumor growthshortly after the treatment period than buffer control (p<0.0001),Cisplatin treatment alone (p=0.004) or H460-16-2 treatment alone(p<0.0001). At the end of the treatment phase, mice given H460-16-2 plusCisplatin had tumors that grew to only 28.5 percent of the buffercontrol group. For PC-3 SCID xenograft models, body weight can be usedas a surrogate indicator of disease progression. Mice in all the groupsexperienced severe weight loss. In this study, mice in all groups showeda weight loss of approximately 23 to 35 percent by the end of thetreatment period. The group treated with H460-16-2 showed the smallestdegree of weight loss (21.7 percent). After treatment, day 48, there wasno significant increase in weight loss associated with the treatment ofH460-16-2 and Cisplatin in comparison to buffer control (p=0.5042).Thus, H460-16-2 plus Cisplatin treatment was efficacious as it delayedtumor growth compared to the isotype control treated group in awell-established model of human prostate cancer.

In order to validate the H460-16-2 epitope as a drug target, theexpression of H460-16-2 antigen in normal human tissues was previouslydetermined (Ser. No. 10/603,000). This work was extended by comparisonwith the anti-CD44 antibodies; clone L178 (disclosed in Ser. No.10/647,818) and clone BU75 (disclosed in Ser. No. 10/810,165). By IHCstaining with H460-16-2, the majority of the tissues failed to expressthe H460-16-2 antigen, including the cells of the vital organs, such asthe liver, kidney (except for marginal staining of tubular epithelialcells), heart, and lung. Results from tissue staining indicated thatH460-16-2 showed restricted binding to various cell types but hadbinding to infiltrating macrophages, lymphocytes, and fibroblasts. TheBU75 antibody showed a similar staining pattern. However, there was atleast one difference of note; staining of lymphocytes was more intensewith BU75 in comparison to H460-16-2.

Localization of the H460-16-2 antigen and determining its prevalencewithin the population, such as among breast cancer patients, isimportant in assessing the therapeutic use of H460-16-2 and designingeffective clinical trials. To address H460-16-2 antigen expression inbreast tumors from cancer patients, tumor tissue samples from 50individual breast cancer patients were previously screened forexpression of the H460-16-2 antigen (Ser. No. 10/603,000) and wascompared to L178 (Ser. No. 10/647,818), BU75 (Ser. No. 10/810,165) andthe anti-Her2 antibody c-erbB-2 (Ser. No. 10/810,165). The results ofthese studies were similar and showed that 62 percent of tissue samplesstained positive for the H460-16-2 antigen while 73 percent of breasttumor tissues were positive for the BU75 epitope. Expression ofH460-16-2 within patient samples appeared specific for cancer cells asstaining was restricted to malignant cells. H460-16-2 stained 4 of 10samples of normal tissue from breast cancer patients while BU75 stained8. Breast tumor expression of both the H460-16-2 and BU75 antigenappeared to be mainly localized to the cell membrane of malignant cells,making CD44 an attractive target for therapy. H460-16-2 expression wasfurther evaluated based on breast tumor expression of the receptors forthe hormones estrogen and progesterone, which play an important role inthe development, treatment, and prognosis of breast tumors. Nocorrelation was apparent between expression of the H460-16-2 antigen andexpression of the receptors for either estrogen or progesterone. Whentumors were analyzed based on their stage, or degree to which the canceradvanced, again there was no clear correlation between H460-16-2 antigenexpression and tumor stage. Similar results were obtained with BU75. Incomparison to c-erbB-2, H460-16-2 showed a completely different stainingprofile where 52 percent of the breast tumor tissue samples that werepositive for the H460-16-2 antigen were negative for Her2 expressionindicating a yet unmet targeted therapeutic need for breast cancerpatients. There were also differences in the intensity of stainingbetween the breast tumor tissue sections that were positive for bothH460-16-2 and Her2. The c-erbB-2 antibody also positively stained one ofthe normal breast tissue sections.

To further extend the potential therapeutic benefit of H460-16-2, thefrequency and localization of the antigen within various human cancertissues was also previously determined (Ser. No. 10/603,000) and wascompared to clone L178 (Ser. No. 10/647,818). The majority of thesetumor types were also positive for the L178 antigen. As with humanbreast tumor tissue, H460-16-2 and L178 localization occurred on themembrane of tumor cells. However, there was substantially more membranelocalization with the L178 compared to the H460-16-2 antibody. Also, ofthe tumor types that were stained by both H460-16-2 and L178, 43 percentof the tissues showed higher intensity staining with the L178 antibody.

In addition, the frequency and localization of the antigen withinprostate and liver normal and cancer tissues was previously determined(Ser. No. 11/364,013). From the prostate cancer array, 19/53 (36percent) of the tested tumors were positive for H460-16-2. H460-16-2 wasspecific for tumor cells and stroma fibroblasts. Cellular localizationwas mostly membranous and cytoplasmic membranous with or without luminallocalization. The percentage of positive cells ranged from <10percent->50 percent indicating heterogenous binding of the antibody totumor cells. The relation of the antibody binding to tumors' stagescould not be assessed properly due to a discrepancy in the number oftumors among different tumor stages, being 1/1 (100 percent), 4/12 (33percent), 0/2 (0 percent) and 11/33 (33 percent) to stage I, II, III andIV, respectively. There was higher binding to Gleason score G3-G4 (36percent) than to G1-G2 (25 percent). The Gleason score is a system ofgrading prostate cancer. The Gleason grading system assigns a grade toeach of the two largest areas of cancer in the tissue samples. Gradesrange from 1 to 5 with 1 being the least aggressive and 5 the mostaggressive. Grade 3 tumors, for example, seldom have metastases, butmetastases are common with grade 4 or grade 5. The two grades are thenadded together to produce a Gleason score. A score of 2 to 4 isconsidered low grade; 5 through 7, intermediate grade; and 8 through 10,high grade. A tumor with a low Gleason score typically grows slowlyenough that it may not pose a significant threat to the patient in hislifetime. All 3 normal prostate tissue sections were positive for theantibody. However, the tissue specificity was for myoepithelium andstromal fibroblasts and spared the glandular epithelium. FIG. 12demonstrates the heterogeneity of the binding of H460-16-2 to testedprostate tumors: 10/53, 6/53, 3/53 positive tumors were in thecategories of <10-10 percent, <50-50 percent and >50 percent,respectively. As a result of its binding to prostate cancer cells, thetherapeutic benefit of H460-16-2 can potentially be extended to thetreatment of prostate cancer.

From the liver cancer array, H460-16-2 antibody showed binding to 21/49(43 percent) of tested liver cancers, including 11/37 (30 percent) ofprimary, 7/8 (88 percent) of metastatic hepatocellular carcinoma, 1/2(50 percent) of primary and 2/2 (100 percent) of metastaticcholangiocarcinomas. The antibody showed significant higher binding toadvanced tumors' stages III and IV in comparison with early stages I andII (p=0.03) [stage I, 0/2 (0 percent); stage II, 2/17 (12 percent);stage III, 8/16 (50 percent) and stage 1V, 6/8 (75 percent)]. H460-16-2was specific for tumor cells and infiltrating inflammatory cells.Cellular localization was mainly membranous. Some tumors also displayeda diffuse cytoplasmic staining pattern. The antibody bound to 9/9 ofnon-neoplastic liver tissues. However, the binding was restricted to thesinusoidal cells and infiltrating lymphocytes. The H460-16-2 antigenappears to be specifically expressed on advanced liver tumor tissue.H460-16-2 therefore has potential as a therapeutic drug in the treatmentof liver cancer.

There appears to be no form of CD44 that exactly matches the IHC datapresented herein based on comparisons with the IHC data from theliterature. The standard form of CD44 is normally expressed in the humanbrain; the H460-16-2 antigen is not. Antibodies directed againstpan-CD44 isoforms do not stain the liver (including Kuppfer cells) andpositively stain the endometrial glands in all phases of thereproductive cycle. The H460-16-2 antigen is clearly present on Kuppfercells and is only present on the secretory endometrial glands of thereproductive cycle. H460-16-2 antigen is clearly present on tissuemacrophages and only the variant forms V4/5 and V8/9 show occasionalmacrophage staining. The similar yet distinct binding pattern seen withH460-16-2 in comparison to anti-CD44 L178 and now BU75 indicates thatthe H460-16-2 antigen is an unique epitope of CD44.

As disclosed previously (Ser. No. 10/647,818), additional biochemicaldata also indicated that the antigen recognized by H460-16-2 is one ofthe forms of CD44. This was supported by studies that showed amonoclonal antibody (L178) reactive against CD44 identifies proteinsthat were bound to H460-16-2 by immunoprecipitation. Western blottingstudies also suggested that the epitope of CD44 recognized by H460-16-2was not present on v6 or v10. The H460-16-2 epitope was alsodistinguished by being carbohydrate and conformation dependent, whereasmany anti-CD44 antibodies are directed against peptide portions of CD44.These IHC and biochemical results demonstrated that H460-16-2 binds to avariant of the CD44 antigen. Thus, the preponderance of evidence showedthat H460-16-2 mediates anti-cancer effects through ligation of anunique carbohydrate dependent conformational epitope present on avariant of CD44. For the purpose of this invention, said epitope isdefined as a “CD44 antigenic moiety” characterized by its ability tobind with a monoclonal antibody encoded by the hybridoma cell lineH460-16-2, antigenic binding fragments thereof or antibody conjugatesthereof.

In order to further elucidate the mechanism behind H460-16-2'santi-cancer effects, hyaluronic acid (HA) binding assays were performed(as disclosed in Ser. No. 10/810,165). It was determined that an averageconcentration of 1.87 (+/−1.01) micrograms/mL of H460-16-2 was requiredto inhibit adhesion of MDA-MB-231 cells to HA by 50 percent. Theseresults indicated that H460-16-2 interacts with, at least in part, theregion(s) on CD44 that are responsible for binding to HA andconsequently could be mediating its anti-cancer effects through downregulation of angiogenesis or tumor invasiveness through the ECM.

In addition to the HA binding assays, a cell cycling experiment wasperformed in order to determine if the H460-16-2 in vitro and in vivoanti-cancer effects were due to regulation of the cell cycle (asdisclosed in Ser. No. 10/810,165). After 24 hours and with 20micrograms/mL of H460-16-2, there was an increase in the number ofMDA-MB-231 apoptotic cells in comparison to the isotype control. Thiseffect also appeared to be dose dependent. Therefore, the efficacy ofH460-16-2 might be also due, in whole or in part, to its apoptoticinducing capabilities.

To further elucidate the mechanism of action for H460-16-2, the effectof H460-16-2 treatment upon apoptosis in MDA-MB-231 tumors grown in vivoin a xenograft model of breast cancer was investigated (as disclosed inSer. No. 11/364,013). Apoptotic cells were counted using morphologicalcriteria such as deletion of single cells, cell shrinkage and compactionof chromatin into a dense mass. The buffer control treatment groupyielded an average total score of 17 cells (±5.29) while the H460-16-2treated group yielded an average total score of 22.5 cells (±4.20).Therefore, there is a trend towards increased apoptosis with H460-16-2treatment as determined using cellular morphology.

Two chimeric versions of H460-16-2 were generated as disclosed in Ser.No. 11/364,013. One version is of isotype IgG1, kappa((ch)ARH460-16-2-IgG1) and the other is of isotype IgG2, kappa((ch)ARH460-16-2-IgG2). Supernatants from chimeric IgG1 and chimericIgG2-secreting clones were able to detect CD44 in a Western blot, with asignal that was similar to that obtained with the murine H460-16-2 (asdisclosed in Ser. No. 11/364,013).

Both of the chimeric antibodies were compared to the murine version ofH460-16-2 in an established model of breast cancer (as disclosed in Ser.No. 11/364,013). Both murine H460-16-2 and (ch)ARH460-16-2-IgG1 reducedtumor growth in an established MDA-MB-231 in vivo model of human breastcancer. At day 62, 5 days after the last dose was administered,treatment with H460-16-2 resulted in a tumor growth inhibition of 39percent (Mean T/C=57 percent). This reduction in tumor growth wassignificantly different from the control (p=0.0037). The chimericantibody (ch)ARH460-16-2-IgG1 resulted in an enhanced tumor growthinhibition (TGI) of 64 percent (Mean T/C=26.9 percent; p<0.0001). Bycontrast, the IgG2 version of the chimeric antibody,(ch)ARH460-16-2-IgG2 showed no inhibition in tumor growth when comparedwith the buffer control (TGI=0 percent; Mean T/C=122 percent; p=0.7264).

Annexin-V staining was performed to determine whether the chimericversions of H460-16-2 were able to induce apoptosis in the same manneras the murine counterpart on the MDA-MB-231 human breast cancer cellline (as disclosed in Ser. No. 11/364,013). Spontaneous apoptoticeffects of cells treated with isotype control were found to be similarto cells treated with vehicle only. The murine and human chimeric IgG1and IgG2 H460-16-2 antibodies were all found to induce apoptosis in thebreast cancer cell line in a dose dependent manner in each experiment,with greater apoptotic effect seen with both the (ch)ARH460-16-2 IgG1and IgG2 antibodies. Results indicate that in vitro the(ch)ARH460-16-2-IgG2 antibody has the greatest apoptotic effect whencompared to the chimeric IgG1 antibody. All 3 antibodies showed anincrease in the percentage necrotic and necrotic/apoptotic populationsover their prospective isotype controls. The largest increase in thepercentage necrotic and necrotic/apoptotic populations was seen with(ch)ARH460-16-2-IgG2, then (ch)ARH460-16-2-IgG1 and then H460-16-2.

In toto, this data demonstrates that the murine and chimeric H460-16-2antigen is a cancer associated antigen and is expressed in humans, andis a pathologically relevant cancer target. Further, this data alsodemonstrates the binding of the H460-16-2 antibody to human cancertissues, and can be used appropriately for assays that can bediagnostic, predictive of therapy, or prognostic. In addition, the cellmembrane localization of this antigen is indicative of the cancer statusof the cell due to the lack of expression of the antigen in mostnon-malignant cells, and this observation permits the use of thisantigen, its gene or derivatives, its protein or its variants to be usedfor assays that can be diagnostic, predictive of therapy, or prognostic.

Other studies, involving the use of anti-CD44 antibodies, havelimitations of therapeutic potential that are not exhibited byH460-16-2. H460-16-2 demonstrates both in vitro and in vivo anti-tumoractivity. Previously described antibodies such as MAK<CD44>M-1.1.12,MAK<CD44>M-2.42.3 and MAK<CD44>M-4.3.16 have no in vitro or in vivocytotoxicity ascribed to them and VFF-18 and Mab U36 show no intrinsictumor cytotoxicity. In addition other anti-CD44 antibodies that haveshown in vivo tumor effects also have certain limitations that are notevident with 1-1460-16-2. For example, ASML1.1, K926, anti-CD44s andIM-78.1 show in vivo anti-tumor activity against rat, murine, rat andmurine tumors grown in xenograft models respectively. H460-16-2demonstrates anti-tumor activity in a model of human cancer. H460-16-2is also directed against human CD44 while antibodies such as ASML1.1recognize only rat CD44. The clone 515 anti-CD44 antibody does inhibitperitoneal tumor implantation of a human ovarian cell line but does notprevent or inhibit tumor growth. H460-16-2 is capable of inhibitinghuman breast tumor growth in a SCID mouse xenograft model. GKW.A3 is ananti-human CD44 monoclonal antibody capable of inhibiting tumor growthof a human metastasizing melanoma grown in mice in a preventative butnot an established model. H460-16-2 has demonstrated significantanti-tumor activity in both preventative and established murinexenograft models of human breast cancer. Consequently, it is quiteapparent that H460-16-2 has superior anti-tumor properties in comparisonto previously described anti-CD44 antibodies. It has demonstrated bothin vitro and in vivo anti-tumor activity on a human breast tumor in SCIDmice and is directed against human CD44. It also exhibits activity in apreventative and established (more clinically relevant) model of humanbreast cancer and it exhibits activity with Cisplatin in an establishedmodel of human prostate cancer.

The present invention describes the development and use of H460-16-2developed by the process described in U.S. Pat. No. 6,180,357 andidentified by, its effect, in a cytotoxic assay, in tumor growth inanimal models and in prolonging survival time in those suffering fromcancerous disease.

This invention represents an advance in the field of cancer treatment inthat it describes reagents that bind specifically to an epitope orepitopes present on the target molecule, CD44, and that directly mediateinhibition of tumor growth and metastasis in in vivo models of humanliver cancer. The preponderance of evidence, disclosed herein,demonstrates that chimeric H460-16-2 mediates anti-cancer effectsthrough ligation of epitopes present on CD44, which is expressed onliver cancer, which will broadly be understood to encompass any primaryor metastatic tumor sites which arise from hepatocytes. This applicationdemonstrates that expression of CD44 is observed with metastatic versusprimary human liver cancer cell lines. This invention also disclosesthat chimeric H460-16-2 reduces the tumor burden and probability ofmetastasis of human liver cancer in vivo.

This is an advance in relation to any other previously describedanti-CD44 antibody, since none have been shown to have similarproperties. It also provides an advance in the field since it clearlydemonstrates the direct involvement of CD44 in events associated withgrowth and development of certain types of tumors. It also represents anadvance in cancer therapy since it has the potential to display similaranti-cancer properties in human patients. A further advance is thatinclusion of these antibodies in a library of anti-cancer antibodieswill enhance the possibility of targeting tumors expressing differentantigen markers by determination of the appropriate combination ofdifferent anti-cancer antibodies, to find the most effective intargeting and inhibiting growth and development of the tumors.

In all, this invention teaches the use of the H460-16-2 antigen as atarget for a therapeutic agent, that when administered can reduce thetumor burden of a cancer expressing the antigen in a mammal (thusdelaying disease progression) and the likelihood of metastasis of acancer expressing the antigen in a mammal, and can also lead to aprolonged survival of the treated mammal. This invention also teachesthe use of a CDMAB (H460-16-2), and its derivatives, ligands and antigenbinding fragments thereof, to target its antigen to reduce the tumorburden of a cancer expressing the antigen in a mammal, and to prolongthe survival of a mammal bearing tumors that express this antigen.Furthermore, this invention also teaches the use of detecting theH460-16-2 antigen in cancerous cells that can be useful for thediagnosis, prediction of therapy, and prognosis of mammals bearingtumors that express this antigen.

Accordingly, it is an objective of the invention to utilize a method forproducing cancerous disease modifying antibodies (CDMAB) raised againstcancerous cells derived from a particular individual, or one or moreparticular cancer cell lines, which CDMAB are cytotoxic with respect tocancer cells while simultaneously being relatively non-toxic tonon-cancerous cells, in order to isolate hybridoma cell lines and thecorresponding isolated monoclonal antibodies and antigen bindingfragments thereof for which said hybridoma cell lines are encoded.

It is an additional objective of the invention to teach cancerousdisease modifying antibodies, ligands and antigen binding fragmentsthereof.

It is a further objective of the instant invention to produce cancerousdisease modifying antibodies whose cytotoxicity is mediated throughantibody dependent cellular toxicity.

It is yet an additional objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is mediatedthrough complement dependent cellular toxicity.

It is still a further objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is a functionof their ability to catalyze hydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to producecancerous disease modifying antibodies which are useful for in a bindingassay for diagnosis, prognosis, and monitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein are set forth, by way of illustrationand example, certain embodiments of this invention.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a summary of H460-16-2 binding on a human liver tumor andnormal tissue microarray.

FIG. 2. Representative micrographs showing the binding pattern on livertumor tissue obtained with H460-16-2 (A) or the isotype control antibody(B) and on non-neoplastic liver tissue obtained with H460-16-2 (C) orthe isotype control antibody (D) from a human tissue microarray.H460-16-2 displayed strong positive staining for the tumor cells and waslimited to staining on sinusoidal cells (black arrows) and infiltratinglymphocytes (green arrows) on the non-neoplastic liver tissue.Magnification is 200×.

FIG. 3 demonstrates the correlation between CD44 over-expression and themetastatic potential of various HCC cell lines.

FIG. 4 demonstrates the effect of (ch)ARH460-16-2-IgG1 on HCC tumorgrowth and metastasis in an established orthotopic HCC tumor model. Datapoints represent the mean+/−SEM.

FIG. 5 demonstrates the quantitative effect of (ch)ARH460-16-2-IgG1 ontumor growth in an established orthotopic HCC tumor model. The bar graphsummarizes the average basal signal of tumors from four groups ofanimals in photons/s/cm²/steridian. Each column represents the averagebasal level signals on day 45 following tumor inoculation.

FIG. 6 visually demonstrates the effect of (ch)ARH460-16-2-IgG1 onprimary tumor growth in an established orthotopic HCC tumor model.

FIG. 7 is the tabulation of the number of different metastasis thatdeveloped with and without antibody treatment.

DETAILED DESCRIPTION OF THE INVENTION

In general, the following words or phrases have the indicated definitionwhen used in the summary, description, examples, and claims.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies, de-immunized, murine, chimericor humanized antibodies), antibody compositions with polyepitopicspecificity, single-chain antibodies, diabodies, triabodies,immunoconjugates and antibody fragments (see below).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma (murine orhuman) method first described by Kohler et al., Nature, 256:495 (1975),or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), for example.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include less than full length antibodies,Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; single-chain antibodies, single domainantibody molecules, fusion proteins, recombinant proteins andmultispecific antibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and Fcγ RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., Eur. J. Immunol. 24:2429 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues2632 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fe” fragment, whose name reflects its ability to crystallize readily.Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-bindingsites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site. The Fab fragment alsocontains the constant domain of the light chain and the first constantdomain (CH I) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear at least onefree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

The term “triabodies” or “trivalent trimers” refers to the combinationof three single chain antibodies. Triabodies are constructed with theamino acid terminus of a V_(L) or V_(H) domain, i.e., without any linkersequence. A triabody has three Fv heads with the polypeptides arrangedin a cyclic, head-to-tail fashion. A possible conformation of thetriabody is planar with the three binding sites located in a plane at anangle of 120 degrees from one another. Triabodies can be monospecific,bispecific or trispecific.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An antibody “which binds” an antigen of interest, e.g. CD44 antigenicmoiety, is one capable of binding that antigen with sufficient affinitysuch that the antibody is useful as a therapeutic or diagnostic agent intargeting a cell expressing the antigen. Where the antibody is one whichbinds CD44 antigenic moiety it will usually preferentially bind CD44antigenic moiety as opposed to other receptors, and does not includeincidental binding such as non-specific Fc contact, or binding topost-translational modifications common to other antigens and may be onewhich does not significantly cross-react with other proteins. Methods,for the detection of an antibody that binds an antigen of interest, arewell known in the art and can include but are not limited to assays suchas FACS, cell ELISA and Western blot.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably, and all such designations include progeny. Itis also understood that all progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included. It will be clear from thecontext where distinct designations are intended.

“Treatment or treating” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented. Hence, the mammal to be treated herein may have beendiagnosed as having the disorder or may be predisposed or susceptible tothe disorder.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth or death. Examples of cancer include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carnomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, mice, SCID or nude mice or strains of mice,domestic and farm animals, and zoo, sports, or pet animals, such assheep, dogs, horses, cats, cows, etc. Preferably, the mammal herein ishuman.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032, published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14:5399-5407, 1986. They are thenpurified on polyacrylamide gels.

In accordance with the present invention, “humanized” and/or “chimeric”forms of non-human (e.g. murine) immunoglobulins refer to antibodieswhich contain specific chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which results in thedecrease of a human anti-mouse antibody (HAMA), human anti-chimericantibody (HACA) or a human anti-human antibody (HAHA) response, comparedto the original antibody, and contain the requisite portions (e.g.CDR(s), antigen binding region(s), variable domain(s) and so on) derivedfrom said non-human immunoglobulin, necessary to reproduce the desiredeffect, while simultaneously retaining binding characteristics which arecomparable to said non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from the complementarity determining regions (CDRs) ofthe recipient antibody are replaced by residues from the CDRs of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human FR residues. Furthermore, the humanizedantibody may comprise residues which are found neither in the recipientantibody nor in the imported CDR or FR sequences. These modificationsare made to further refine and optimize antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR residues are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

“De-immunized” antibodies are immunoglobulins that are non-immunogenic,or less immunogenic, to a given species. De-immunization can be achievedthrough structural alterations to the antibody. Any de-immunizationtechnique known to those skilled in the art can be employed. Onesuitable technique for de-immunizing antibodies is described, forexample, in WO 00/34317 published Jun. 15, 2000.

An antibody which induces “apoptosis” is one which induces programmedcell death by any means, illustrated by but not limited to binding ofannexin V, caspase activity, fragmentation of DNA, cell shrinkage,dilation of endoplasmic reticulum, cell fragmentation, and/or formationof membrane vesicles (called apoptotic bodies).

As used herein “antibody induced cytotoxicity” is understood to mean thecytotoxic effect derived from the hybridoma supernatant or antibodyproduced by the hybridoma deposited with the ATCC as accession numberPTA-4621, a humanized antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as accession numberPTA-4621, a chimeric antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as accession numberPTA-4621, antigen binding fragments, or antibody ligands thereof, whicheffect is not necessarily related to the degree of binding.

Throughout the instant specification, hybridoma cell lines, as well asthe isolated monoclonal antibodies which are produced therefrom, arealternatively referred to by their internal designation, H460-16-2(murine), (h)ARH460-16-2-IgG1, (ch)ARH460-16-2-IgG1, or DepositoryDesignation, ATCC PTA-4621.

As used herein “antibody-ligand” includes a moiety which exhibitsbinding specificity for at least one epitope of the target antigen, andwhich may be an intact antibody molecule, antibody fragments, and anymolecule having at least an antigen-binding region or portion thereof(i.e., the variable portion of an antibody molecule), e.g., an Fvmolecule, Fab molecule, Fab′ molecule, F(ab)₂ molecule, a bispecificantibody, a fusion protein, or any genetically engineered molecule whichspecifically recognizes and binds at least one epitope of the antigenbound by the isolated monoclonal antibody produced by the hybridoma cellline designated as ATCC PTA-4621 (the ATCC PTA-4621 antigen), ahumanized antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621, achimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621, andantigen binding fragments thereof.

As used herein “cancerous disease modifying antibodies” (CDMAB) refersto monoclonal antibodies which modify the cancerous disease process in amanner which is beneficial to the patient, for example by reducing tumorburden or prolonging survival of tumor bearing individuals, andantibody-ligands thereof.

A “CDMAB related binding agent”, in its broadest sense, is understood toinclude, but is not limited to, any form of human or non-humanantibodies, antibody fragments, antibody ligands, or the like, whichcompetitively bind to at least one CDMAB target epitope.

A “competitive binder” is understood to include any form of human ornon-human antibodies, antibody fragments, antibody ligands, or the likewhich has binding affinity for at least one CDMAB target epitope.

Tumors to be treated include primary tumors and metastatic tumors, aswell as refractory tumors. Refractory tumors include tumors that fail torespond or are resistant to treatment with chemotherapeutic agentsalone, antibodies alone, radiation alone or combinations thereof.Refractory tumors also encompass tumors that appear to be inhibited bytreatment with such agents but recur up to five years, sometimes up toten years or longer after treatment is discontinued.

Tumors that can be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors.Examples of solid tumors, which can be accordingly treated, includebreast carcinoma, lung carcinoma, colorectal carcinoma, pancreaticcarcinoma, glioma and lymphoma. Some examples of such tumors includeepidermoid tumors, squamous tumors, such as head and neck tumors,colorectal tumors, prostate tumors, breast tumors, lung tumors,including small cell and non-small cell lung tumors, pancreatic tumors,thyroid tumors, ovarian tumors, and liver tumors. Other examples includeKaposi's sarcoma, CNS neoplasms, neuroblastomas, capillaryhemangioblastomas, meningiomas and cerebral metastases, melanoma,gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma,glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.

As used herein “antigen-binding region” means a portion of the moleculewhich recognizes the target antigen.

As used herein “competitively inhibits” means being able to recognizeand bind a determinant site to which the monoclonal antibody produced bythe hybridoma cell line designated as ATCC PTA-4621 (the ATCC PTA-4621antibody), a humanized antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as accession numberPTA-4621, a chimeric antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as accession numberPTA-4621, antigen binding fragments, or antibody ligands thereof, isdirected using conventional reciprocal antibody competition assays.(Belanger L., Sylvestre C. and Dufour D. (1973), Enzyme linkedimmunoassay for alpha fetoprotein by competitive and sandwichprocedures. Clinica Chimica Acta 48, 15).

As used herein “target antigen” is the ATCC PTA-4621 antigen or portionsthereof.

As used herein, an “immunoconjugate” means any molecule or CDMAB such asan antibody chemically or biologically linked to cytotoxins, radioactiveagents, cytokines, interferons, target or reporter moieties, enzymes,toxins, anti-tumor drugs or therapeutic agents. The antibody or CDMABmay be linked to the cytotoxin, radioactive agent, cytokine, interferon,target or reporter moiety, enzyme, toxin, anti-tumor drug or therapeuticagent at any location along the molecule so long as it is able to bindits target. Examples of immunoconjugates include antibody toxin chemicalconjugates and antibody-toxin fusion proteins.

Radioactive agents suitable for use as anti-tumor agents are known tothose skilled in the art. For example, 131I or 211At is used. Theseisotopes are attached to the antibody using conventional techniques(e.g. Pedley et al., Br. J. Cancer 68, 69-73 (1993)). Alternatively, theanti-tumor agent which is attached to the antibody is an enzyme whichactivates a prodrug. A prodrug may be administered which will remain inits inactive form until it reaches the tumor site where it is convertedto its cytotoxin form once the antibody complex is administered. Inpractice, the antibody-enzyme conjugate is administered to the patientand allowed to localize in the region of the tissue to be treated. Theprodrug is then administered to the patient so that conversion to thecytotoxic drug occurs in the region of the tissue to be treated.Alternatively, the anti-tumor agent conjugated to the antibody is acytokine such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumornecrosis factor alpha (TNF-α). The antibody targets the cytokine to thetumor so that the cytokine mediates damage to or destruction of thetumor without affecting other tissues. The cytokine is fused to theantibody at the DNA level using conventional recombinant DNA techniques.Interferons may also be used.

As used herein, a “fusion protein” means any chimeric protein wherein anantigen binding region is connected to a biologically active molecule,e.g., toxin, enzyme, fluorescent proteins, luminescent marker,polypeptide tag, cytokine, interferon, target or reporter moiety orprotein drug.

The invention further contemplates CDMAB of the present invention towhich target or reporter moieties are linked. Target moieties are firstmembers of binding pairs. Anti-tumor agents, for example, are conjugatedto second members of such pairs and are thereby directed to the sitewhere the antigen-binding protein is bound. A common example of such abinding pair is avidin and biotin. In a preferred embodiment, biotin isconjugated to the target antigen of the CDMAB of the present invention,and thereby provides a target for an anti-tumor agent or other moietywhich is conjugated to avidin or streptavidin. Alternatively, biotin oranother such moiety is linked to the target antigen of the CDMAB of thepresent invention and used as a reporter, for example in a diagnosticsystem where a detectable signal-producing agent is conjugated to avidinor streptavidin.

Detectable signal-producing agents are useful in vivo and in vitro fordiagnostic purposes. The signal producing agent produces a measurablesignal which is detectable by external means, usually the measurement ofelectromagnetic radiation. For the most part, the signal producing agentis an enzyme or chromophore, or emits light by fluorescence,phosphorescence or chemiluminescence. Chromophores include dyes whichabsorb light in the ultraviolet or visible region, and can be substratesor degradation products of enzyme catalyzed reactions.

Moreover, included within the scope of the present invention is use ofthe present CDMAB in vivo and in vitro for investigative or diagnosticmethods, which are well known in the art. In order to carry out thediagnostic methods as contemplated herein, the instant invention mayfurther include kits, which contain CDMAB of the present invention. Suchkits will be useful for identification of individuals at risk forcertain type of cancers by detecting over-expression of the CDMAB'starget antigen on cells of such individuals.

Diagnostic Assay Kits

It is contemplated to utilize any suitable CDMAB in accordance with thepresent invention in the form of a diagnostic assay kit for determiningthe presence of a tumor. The tumor will generally be detected in apatient based on the presence of one or more tumor-specific antigens,e.g. proteins and/or polynucleotides which encode such proteins in abiological sample, such as blood, sera, urine and/or tumor biopsies,which samples will have been obtained from the patient.

The proteins function as markers which indicate the presence or absenceof a particular tumor, for example a colon, breast, lung or prostatetumor. It is further contemplated that the antigen will have utility forthe detection of other cancerous tumors. Inclusion in the diagnosticassay kits of binding agents comprised of CDMABs of the presentinvention, or CDMAB related binding agents, enables detection of thelevel of antigen that binds to the agent in the biological sample.Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a tumor protein, which is also indicative of the presenceor absence of a cancer. In order for the binding assay to be diagnostic,data will have been generated which correlates statistically significantlevels of antigen, in relation to that present in normal tissue, so asto render the recognition of binding definitively diagnostic for thepresence of a cancerous tumor. It is contemplated that a plurality offormats will be useful for the diagnostic assay of the presentinvention, as are known to those of ordinary skill in the art, for usinga binding agent to detect polypeptide markers in a sample. For example,as illustrated in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Chapters 9-14, 1988. Further contemplated areany and all combinations, permutations or modifications of theafore-described diagnostic assay formats.

The presence or absence of a cancer in a patient will typically bedetermined by (a) contacting a biological sample obtained from a patientwith a binding agent; (b) detecting in the sample a level of polypeptidethat binds to the binding agent; and (c) comparing the level ofpolypeptide with a predetermined cut-off value.

In an illustrative embodiment, it is contemplated that the assay willinvolve the use of a CDMAB based binding agent immobilized on a solidsupport to bind to and remove the polypeptide from the remainder of thesample. The bound polypeptide may then be detected using a detectionreagent that contains a reporter group and specifically binds to thebinding agent/polypeptide complex. Illustrative detection reagents mayinclude a CDMAB based binding agent that specifically binds to thepolypeptide or an antibody or other agent that specifically binds to thebinding agent, such as an anti-immunoglobulin, protein G, protein A or alectin. In an alternative embodiment, it is contemplated that acompetitive assay may be utilized, in which a polypeptide is labeledwith a reporter group and allowed to bind to the immobilized bindingagent after incubation of the binding agent with the sample. Indicativeof the reactivity of the sample with the immobilized binding agent, isthe extent to which components of the sample inhibit the binding of thelabeled polypeptide to the binding agent. Suitable polypeptides for usewithin such assays include full length tumor-specific proteins and/orportions thereof, to which the binding agent has binding affinity.

The diagnostic kit will be provided with a solid support which may be inthe form of any material known to those of ordinary skill in the art towhich the protein may be attached. Suitable examples may include a testwell in a microtiter plate or a nitrocellulose or other suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. The support may also be a magnetic particle or afiber optic sensor, such as those disclosed, for example, in U.S. Pat.No. 5,359,681.

It is contemplated that the binding agent will be immobilized on thesolid support using a variety of techniques known to those of skill inthe art, which are amply described in the patent and scientificliterature. The term “immobilization” refers to both noncovalentassociation, such as adsorption, and covalent attachment, which, in thecontext of the present invention, may be a direct linkage between theagent and functional groups on the support, or may be a linkage by wayof a cross-linking agent. In a preferred, albeit non-limitingembodiment, immobilization by adsorption to a well in a microtiter plateor to a membrane is preferable. Adsorption may be achieved by contactingthe binding agent, in a suitable buffer, with the solid support for asuitable amount of time. The contact time may vary with temperature, andwill generally be within a range of between about 1 hour and about 1day.

Covalent attachment of binding agent to a solid support would ordinarilybe accomplished by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the binding agent. For example,the binding agent may be covalently attached to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe binding partner.

It is further contemplated that the diagnostic assay kit will take theform of a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody, e.g. the instantly disclosed CDMAB thathas been immobilized on a solid support, commonly the well of amicrotiter plate, with the sample, such that polypeptides within thesample are allowed to bind to the immobilized antibody. Unbound sampleis then removed from the immobilized polypeptide-antibody complexes anda detection reagent (preferably a second antibody capable of binding toa different site on the polypeptide) containing a reporter group isadded. The amount of detection reagent that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

In a specific embodiment, it is contemplated that once the antibody isimmobilized on the support as described above, the remaining proteinbinding sites on the support will be blocked, via the use of anysuitable blocking agent known to those of ordinary skill in the art,such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St.Louis, Mo.). The immobilized antibody would then be incubated with thesample, and polypeptide would be allowed to bind to the antibody. Thesample could be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) would be selected tocorrespond to a period of time sufficient to detect the presence ofpolypeptide within a sample obtained from an individual with thespecifically selected tumor. Preferably, the contact time is sufficientto achieve a level of binding that is at least about 95 percent of thatachieved at equilibrium between bound and unbound polypeptide. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time.

It is further contemplated that unbound sample would then be removed bywashing the solid support with an appropriate buffer. The secondantibody, which contains a reporter group, would then be added to thesolid support. Incubation of the detection reagent with the immobilizedantibody-polypeptide complex would then be carried out for an amount oftime sufficient to detect the bound polypeptide. Subsequently, unbounddetection reagent would then be removed and bound detection reagentwould be detected using the reporter group. The method employed fordetecting the reporter group is necessarily specific to the type ofreporter group selected, for example for radioactive groups,scintillation counting or autoradiographic methods are generallyappropriate. Spectroscopic methods may be used to detect dyes,luminescent groups and fluorescent groups. Biotin may be detected usingavidin, coupled to a different reporter group (commonly a radioactive orfluorescent group or an enzyme). Enzyme reporter groups may generally bedetected by the addition of substrate (generally for a specific periodof time), followed by spectroscopic or other analysis of the reactionproducts.

In order to utilize the diagnostic assay kit of the present invention todetermine the presence or absence of a cancer, such as prostate cancer,the signal detected from the reporter group that remains bound to thesolid support would generally be compared to a signal that correspondsto a predetermined cut-off value. For example, an illustrative cut-offvalue for the detection of a cancer may be the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is about three standard deviations above the predetermined cut-offvalue would be considered positive for the cancer. In an alternateembodiment, the cut-off value might be determined by using a ReceiverOperator Curve, according to the method of Sackett, ClinicalEpidemiology. A Basic Science for Clinical Medicine, Little Brown andCo., 1985, p. 106-7. In such an embodiment, the cut-off value could bedetermined from a plot of pairs of true positive rates (i.e.,sensitivity) and false positive rates (100 percent-specificity) thatcorrespond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperleft-hand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate. In general, a samplegenerating a signal that is higher than the cut-off value determined bythis method is considered positive for a cancer.

It is contemplated that the diagnostic assay enabled by the kit will beperformed in either a flow-through or strip test format, wherein thebinding agent is immobilized on a membrane, such as nitrocellulose. Inthe flow-through test, polypeptides within the sample bind to theimmobilized binding agent as the sample passes through the membrane. Asecond, labeled binding agent then binds to the bindingagent-polypeptide complex as a solution containing the second bindingagent flows through the membrane. The detection of bound second bindingagent may then be performed as described above. In the strip testformat, one end of the membrane to which binding agent is bound will beimmersed in a solution containing the sample. The sample migrates alongthe membrane through a region containing second binding agent and to thearea of immobilized binding agent. Concentration of the second bindingagent at the area of immobilized antibody indicates the presence of acancer. Generation of a pattern, such as a line, at the binding site,which can be read visually, will be indicative of a positive test. Theabsence of such a pattern indicates a negative result. In general, theamount of binding agent immobilized on the membrane is selected togenerate a visually discernible pattern when the biological samplecontains a level of polypeptide that would be sufficient to generate apositive signal in the two-antibody sandwich assay, in the formatdiscussed above. Preferred binding agents for use in the instantdiagnostic assay are the instantly disclosed antibodies, antigen-bindingfragments thereof, and any CDMAB related binding agents as hereindescribed. The amount of antibody immobilized on the membrane will beany amount effective to produce a diagnostic assay, and may range fromabout 25 nanograms to about 1 microgram. Typically such tests may beperformed with a very small amount of biological sample.

Additionally, CDMABs of the present invention may be used in thelaboratory for research due to its ability to identify its targetantigen.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The present invention provides CDMABs (i.e., ATCC PTA-4621 CDMAB, ahumanized antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621, achimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621, antigenbinding fragments, or antibody ligands thereof) which specificallyrecognize and bind the ATCC PTA-4621 antigen.

The CDMABs of the isolated monoclonal antibody produced by the hybridomadeposited with the ATCC as accession number PTA-4621 may be in any formas long as it has an antigen-binding region which competitively inhibitsthe immunospecific binding of the isolated monoclonal antibody producedby hybridoma ATCC PTA-4621 to its target antigen. Thus, any recombinantproteins (e.g., fusion proteins wherein the antibody is combined with asecond protein such as a lymphokine or a tumor inhibitory growth factor)having the same binding specificity as the ATCC PTA-4621 antibody fallwithin the scope of this invention.

In one embodiment of the invention, the CDMAB is the ATCC PTA-4621antibody.

In other embodiments, the CDMAB is an antigen binding fragment which maybe a Fv molecule (such as a single-chain Fv molecule), a Fab molecule, aFab′ molecule, a F(ab′)₂ molecule, a fusion protein, a bispecificantibody, a heteroantibody or any recombinant molecule having theantigen-binding region of the ATCC PTA-4621 antibody. The CDMAB of theinvention is directed to the epitope to which the ATCC PTA-4621monoclonal antibody is directed.

The CDMAB of the invention may be modified, i.e., by amino acidmodifications within the molecule, so as to produce derivativemolecules. Chemical modification may also be possible. Modification bydirect mutation, methods of affinity maturation, phage display or chainshuffling may also be possible.

Affinity and specificity can be modified or improved by mutating CDRand/or phenylalanine tryptophan (FW) residues and screening for antigenbinding sites having the desired characteristics (e.g., Yang et al., J.Mol. Biol., (1995) 254: 392-403). One way is to randomize individualresidues or combinations of residues so that in a population ofotherwise identical antigen binding sites, subsets of from two to twentyamino acids are found at particular positions. Alternatively, mutationscan be induced over a range of residues by error prone PCR methods(e.g., Hawkins et al., J. Mol. Biol., (1992) 226: 889-96). In anotherexample, phage display vectors containing heavy and light chain variableregion genes can be propagated in mutator strains of E. coli (e.g., Lowet al., J. Mol. Biol., (1996) 250: 359-68). These methods of mutagenesisare illustrative of the many methods known to one of skill in the art.

Another manner for increasing affinity of the antibodies of the presentinvention is to carry out chain shuffling, where the heavy or lightchain are randomly paired with other heavy or light chains to prepare anantibody with higher affinity. The various CDRs of the antibodies mayalso be shuffled with the corresponding CDRs in other antibodies.

Derivative molecules would retain the functional property of thepolypeptide, namely, the molecule having such substitutions will stillpermit the binding of the polypeptide to the IDAC 051206-01 antigen orportions thereof.

These amino acid substitutions include, but are not necessarily limitedto, amino acid substitutions known in the art as “conservative”.

For example, it is a well-established principle of protein chemistrythat certain amino acid substitutions, entitled “conservative amino acidsubstitutions,” can frequently be made in a protein without alteringeither the conformation or the function of the protein.

Such changes include substituting any of isoleucine (I), valine (V), andleucine (L) for any other of these hydrophobic amino acids; asparticacid (D) for glutamic acid (E) and vice versa; glutamine (Q) forasparagine (N) and vice versa; and serine (S) for threonine (T) and viceversa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanineand valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

Example 1 Human Liver Tumor Tissue Staining

IHC studies were conducted to further evaluate the binding of H460-16-2to human liver tumor tissue. IHC optimization studies were performedpreviously in order to determine the conditions for further experiments.H460-16-2 monoclonal antibodies were produced and purified as previouslydisclosed in Ser. No. 10/603,000.

Binding of antibodies to 49 human liver tumor and 9 normal liver tissueswas performed using a human, liver normal and tumor tissue microarray(Imgenex, San Diego, Calif.). The following information was provided foreach patient: age, sex, organ and diagnosis. Tissue sections weredeparaffinized by drying in an oven at 58° C. for 1 hour and dewaxed byimmersing in xylene 5 times for 4 minutes each in Coplin jars. Followingtreatment through a series of graded ethanol washes (100 percent-75percent) the sections were re-hydrated in water. The slides wereimmersed in 10 mM citrate buffer at pH 6 (Dako, Toronto, Ontario) thenmicrowaved at high, medium, and low power settings for 5 minutes eachand finally immersed in cold PBS. Slides were then immersed in 3 percenthydrogen peroxide solution for 6 minutes, washed with PBS three timesfor 5 minutes each, dried, incubated with Universal blocking solution(Dako, Toronto, Ontario) for 5 minutes at room temperature. H460-16-2,anti-AFP (alpha 1 fetoprotein; clone AFP-11, Abcam, Cambridge, Mass.) orisotype control antibody (directed towards Aspergillus niger glucoseoxidase, an enzyme which is neither present nor inducible in mammaliantissues; Dako, Toronto, Ontario) were diluted in antibody dilutionbuffer (Dako, Toronto, Ontario) to its working concentration (5micrograms/mL for each antibody except for anti-PSMA which was dilutedto 10 micrograms/mL) and incubated for 1 hour at room temperature. Theslides were washed with PBS 3 times for 5 minutes each. Immunoreactivityof the primary antibodies was detected/visualized with HRP conjugatedsecondary antibodies as supplied (Dako Envision System, Toronto,Ontario) for 30 minutes at room temperature. Following this step theslides were washed with PBS 3 times for 5 minutes each and a colorreaction developed by adding DAB (3,3′-diaminobenzidinetetrahydrachloride, Dako, Toronto, Ontario) chromogen substrate solutionfor immunoperoxidase staining for 10 minutes at room temperature.Washing the slides in tap water terminated the chromogenic reaction.Following counterstaining with Meyer's Hematoxylin (Sigma Diagnostics,Oakville, ON), the slides were dehydrated with graded ethanols (75-100percent) and cleared with xylene. Using mounting media (Dako Faramount,Toronto, Ontario) the slides were coverslipped. Slides weremicroscopically examined using an Axiovert 200 (Ziess Canada, Toronto,ON) and digital images acquired and stored using Northern EclipseImaging Software (Mississauga, ON). Results were read, scored andinterpreted by a histopathologist.

As disclosed in FIG. 1, the H460-16-2 antibody showed binding to 21/49(43 percent) of tested liver cancers, including 11/37 (30 percent) ofprimary, 7/8 (88 percent) of metastatic hepatocellular carcinoma, 1/2(50 percent) of primary and 2/2 (100 percent) of metastaticcholangiocarcinomas. The antibody showed significant higher binding toadvanced tumors' stages III and IV in comparison with early stages I andII (p=0.03) [stage I, 0/2 (0 percent); stage II, 2/17 (12 percent);stage III, 8/16 (50 percent) and stage IV, 6/8 (75 percent)]. H460-16-2was specific for tumor cells and infiltrating inflammatory cells.Cellular localization was mainly membranous. Some tumors also displayeda diffuse cytoplasmic staining pattern. The antibody bound to 9/9 ofnon-neoplastic liver tissues (FIG. 2). However, the binding wasrestricted to the sinusoidal cells and infiltrating lymphocytes. TheH460-16-2 antigen appears to be specifically expressed on advanced livertumor tissue. H460-16-2 therefore has potential as a therapeutic drug inthe treatment of liver cancer.

Therefore, the H460-16-2 antigen appears to be expressed on liver tumortissue with binding preference for metastatic and advanced liver tumortissue. H460-16-2 therefore has utility as a diagnostic reagent forhepatocellular carcinoma, and as a therapeutic drug in the treatment ofliver cancer.

Example 2 Correlation of CD44 with Metastatic Potential of Various HCCCell Lines

To further evaluate this correlation in vitro, six heptaocellularcarcinoma (HCC) cell lines with various metastatic potential (Hep3B(American Type Culture Collection, Manassas, Va.), Huh-7 (a gift fromDr. H. Nakabayashi, Hokkaido University School of Medicine, Sapporo,Japan), PLC (Japanese Cancer Research Bank, Tokyo, Japan), MHCC-97L,MHCC-97H and HCCLM3 (Liver Cancer Institute, Fudan University, Shanghai,China)) were evaluated for CD44 expression by flow cytometry using(ch)ARH460-16-2-IgG1 antibody. To detect expression of CD44 in variousHCC cell lines, cells were stained for 1 hour with (ch)ARH460-16-2-IgG1or isotype control antibody (10 micrograms/mL) and 30 minutes with theappropriate secondary antibody and analyzed by FACS.

By flow cytometry, CD44 expression levels were found to be higher in themetastatic HCC cell lines (MHCC-97L, HCCLM3 and MHCC-97H) when comparedwith the primary non-metastatic cell lines (Hep3B, Huh-7 and PLC) (FIG.3).

Example 3 Orthotopic HCC Tumor Model with HCCLM3 Cells LuciferaseLabeling of Cells

To further evaluate this correlation in vivo, (ch)ARH460-16-2-IgG1 wastested in an orthotopic HCC tumor model. For the luciferase labelling ofHCCLM3 (a metastatic HCC cell line Yang et al., Cancer Genet. Cytogenet.158(2):180-183 2005) cells, lentiviral vector harboring the luciferasegene was constructed and transfected into the cells using the methoddescribed previously (Cheung et al., Cancer Res. 66(8):4357-4367 2006).Stable transfectants were generated from a pool of greater than 20positive clones (which were selected with blasticidin at a concentrationof 2 micrograms/mL).

Animal CCD Experiments

One million HCCLM3 luciferase labeled cells were subcutaneously injectedinto the right flank of nude mice, which were then observed daily forsigns of tumor development. Once the tumors reached 1 to 1.5 cm indiameter, it was removed and cut into about 1- to 2-mm cubes, which werethen subsequently implanted into the left liver lobe of 5 week old malenude mice. Ten days later, the nude mice were randomized into a group offour and were treated either with isotype control or 2, 10 or 20 mg/kg(ch)ARH460-16-2-IgG1. (ch)ARH460-16-2-IgG1 test antibody wasadministered intraperitoneally to each cohort, in a volume of 200microlitres after dilution from the stock concentration with a diluentthat contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibodies were then administered 2 times per week for a total of 12doses in the same fashion until day 45 post-implantation.

The mice were imaged on day 7 and day 45 after tumor inoculation. Micewere anesthetized with a ketamine-xylazine mix in a 4:1 ratio accordingto the Committee on the Use of Live Animals in Teaching and Research ofthe University of Hong Kong.

Imaging was done using a Xenogen IVIS 100 cooled CCD camera (Xenogen,New Jersey, USA). The mice were injected with 200 microliters of 15mg/mL D-luciferin intraperitoneally for 15 minutes before imaging afterwhich they were placed in a light-tight chamber. A gray-scale referenceimage was obtained followed by the acquisition of a bioluminescentimage. The acquisition time ranged from 3 seconds to 1 minute. Theimages shown are pseudoimages of the emitted light inphotons/s/cm2/steradian, superimposed over the gray-scale photographs ofthe animal.

(ch)ARH460-16-2-IgG1 significantly reduced tumor burden in anestablished model of human HCC (FIG. 4). On day 45 after tumorimplantation, (ch)ARH460-16-2-IgG1 decreased primary liver tumor signalfrom 37.6E+7±0.17 to 9E+7±0.72, 2.3 E+7±0.52 and 0.1E+7±0.5 at the dosesof 2, 10 and 20 mg/kg, respectively (FIG. 5). Representative primarytumors from the different groups were also photographed (FIG. 6). Therewas no significant difference in mean body weight between the two groupsover the course of the study.

(ch)ARH460-16-2-IgG1 significantly suppressed intrahepatic and lungmetastases in an established orthotopic model of human HCC. The numberof mice with lung and intrahepatic metastases in the treatment group andcontrol group are shown in FIG. 7. (ch)ARH460-16-2-IgG1 significantlysuppressed intrahepatic metastasis from (5/5) 100 percent to (2/5) 40percent at a dose of 2 mg/kg. (ch)ARH460-16-2-IgG1 also significantlysuppressed lung metastasis from (5/5) 100 percent to (0/5) 0 percent ata dose of 2 mg/kg (FIG. 7). Besides the liver and lung metastasis,control groups also showed intestine (4/5) and urinary (3/5) metastasis.

In summary, (ch)ARH460-16-2-IgG1 was well-tolerated, decreased the tumorburden and intrahepatic and lung metastases in this established humanorthotopic HCC tumor model.

The preponderance of evidence shows that murine and chimeric H460-16-2mediates anti-cancer effects through ligation of epitopes present onCD44, which is expressed on liver cancer. It has been shown that higherexpression of CD44 is observed with metastatic versus primary humanliver cancer cell lines. It has also been shown that chimeric H460-16-2reduces the tumor burden and probability of metastasis of human livercancer in vivo. Therefore, chimeric 11460-16-2 has therapeutic potentialfor the diagnosis and treatment of liver cancer, broadly understood toinclude any primary or metastatic tumor sites which arise fromhepatocytes.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Anyoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method of treating primary human tumor sites and metastatic sites,wherein said primary human tumor or metastasis expresses at least oneepitope of an antigen which specifically binds to the isolatedmonoclonal antibody produced by a clone deposited with the ATCC asaccession number PTA-4621 or a CDMAB thereof, which is characterized byan ability to competitively inhibit binding of said isolated monoclonalantibody or CDMAB thereof to its target antigen, comprisingadministering to said mammal said isolated monoclonal antibody or saidCDMAB thereof in an amount effective to result in a reduction of saidmammal's tumor burden.
 2. The method of claim 1 wherein said isolatedmonoclonal antibody or CDMAB thereof is conjugated to a cytotoxicmoiety.
 3. The method of claim 1 wherein said cytotoxic moiety is aradioactive isotope.
 4. The method of claim 1 wherein said isolatedmonoclonal antibody or CDMAB thereof activates complement.
 5. The methodof claim 1 wherein said isolated monoclonal antibody or CDMAB thereofmediates antibody dependent cellular cytotoxicity.
 6. The method ofclaim 1 wherein said isolated monoclonal antibody is a humanizedantibody of the isolated monoclonal antibody produced by the hybridomadeposited with the ATCC as accession number PTA-4621 or a CDMAB thereof.7. The method of claim 1 wherein said isolated monoclonal antibody is achimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621 or aCDMAB thereof.
 8. A method of treating primary human tumor sites andmetastatic sites susceptible to antibody induced cellular cytotoxicityin a mammal, wherein said primary human tumor or metastasis expresses atleast one epitope of an antigen which specifically binds to the isolatedmonoclonal antibody produced by a clone deposited with the ATCC asaccession number PTA-4621 or a CDMAB thereof, which is characterized byan ability to competitively inhibit binding of said isolated monoclonalantibody or CDMAB thereof to its target antigen, comprisingadministering to said mammal said isolated monoclonal antibody or saidCDMAB thereof in an amount effective to result in a reduction of saidmammal's tumor burden.
 9. The method of claim 8 wherein said isolatedmonoclonal antibody or CDMAB thereof is conjugated to a cytotoxicmoiety.
 10. The method of claim 8 wherein said cytotoxic moiety is aradioactive isotope.
 11. The method of claim 8 wherein said isolatedmonoclonal antibody or CDMAB thereof activates complement.
 12. Themethod of claim 8 wherein said isolated monoclonal antibody or CDMABthereof mediates antibody dependent cellular cytotoxicity.
 13. Themethod of claim 8 wherein said isolated monoclonal antibody is ahumanized antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as accession number PTA-4621 or aCDMAB thereof.
 14. The method of claim 8 wherein said isolatedmonoclonal antibody is a chimeric antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the ATCC as accessionnumber PTA-4621 or a CDMAB thereof.
 15. A process for treating humancancerous tumors which express an epitope or epitopes of human CD44antigen which is specifically bound by the isolated monoclonal antibodyproduced by hybridoma cell line H460-16-2 having ATCC Accession No.PTA-4621, comprising: administering to an individual suffering from saidhuman cancer, at least one isolated monoclonal antibody or CDMAB thereofthat recognizes the same epitope or epitopes as those recognized by theisolated monoclonal antibody produced by hybridoma cell line H460-16-2having ATCC Accession No. PTA-4621; wherein binding of said epitope orepitopes results in a reduction of tumor burden.
 16. A process fortreating human cancerous tumors which express an epitope or epitopes ofhuman CD44 antigen which is specifically bound by the isolatedmonoclonal antibody produced by hybridoma cell line H460-16-2 havingATCC Accession No. PTA-4621, comprising: administering to an individualsuffering from said human cancer, at least one isolated monoclonalantibody or CDMAB thereof that recognizes the same epitope or epitopesas those recognized by the isolated monoclonal antibody produced byhybridoma cell line H460-16-2 having ATCC Accession No. PTA-4621;wherein said administration results in a reduction of tumor burden. 17.(canceled)
 18. The process of claim 1, wherein said primary human tumorsites and/or metastatic sites arise from hepatocytes.
 19. The process ofclaim 8, wherein said primary human tumor sites and/or metastatic sitesarise from hepatocytes.
 20. The process of claim 15, wherein said humancancerous tumor arises from hepatocytes.
 21. The process of claim 16,wherein said human cancerous tumor arises from hepatocytes. 22-23.(canceled)